Received: 1 November 2016

|

Revised: 26 February 2017

|

Accepted: 6 March 2017

DOI: 10.1002/btm2.10059

REVIEW

Emerging applications of exosomes in cancer therapeutics and diagnostics Sahil Inamdar1* | Rajeshwar Nitiyanandan2* | Kaushal Rege1 1 Chemical Engineering, Arizona State University, Tempe, AZ 85287

Abstract Exosomes are nanoscale extracellular vesicles that are shed from different cells in the body. Exo-

2 Biological Design Program, Arizona State University, Tempe, AZ 85287

somes encapsulate several biomolecules including lipids, proteins, and nucleic acids, and can therefore play a key role in cellular communication. These vesicles can be isolated from different

Correspondence Kaushal Rege, Chemical Engineering, 501 E. Tyler Mall, ECG 303, Arizona State University, Tempe, AZ 85287. Email: [email protected].

body fluids and their small sizes make them attractive in various biomedical applications. Here, we review state-of-the art approaches in exosome isolation and purification, and describe their potential use in cancer vaccines, drug delivery, and diagnostics. KEYWORDS

Funding information National Science Foundation, Grant/Award Numbers: 1404084 and 1403860; Arizona Biomedical Research Commission, Grant/ Award Number: ADHS14-0829981; National Institutes of Health, Grant/Award Number: 1R01GM093229-01A1

drug delivery, immunotherapies, nanobiology, patient-targeted therapies

nucleic acids, from the cell of origin.10 Proteins including those from

1 | INTRODUCTION

the tetraspanin family (CD9, CD63, CD81, and CD82), ESCRT complex Communication between cells is a critical process that occurs in all

(TSG101, Alix)11 and heat shock proteins (Hsp 60, Hsp70, Hsp90) are

organisms. This sharing of information is facilitated either horizontal

known to be found in exosomes; the composition of these proteins dif-

gene transfer by viruses1 and bacteriophages2 or by the secretion of

fers based on the cell or tissue of origin.12,13

soluble factors.3 Cells secrete extracellular vesicles (EVs), namely, apo4

Vesicles secreted by cells can meet one of the following fates: (a)

ptotic bodies, microvesicles, and exosomes, which can have significant

internalization by other cells in the immediate proximity, (b) internaliza-

impact on the local microenvironment as well as on distant tissues in

tion by cells at a significant distance away from the cell of origin, and

the body.5 Apoptotic bodies are the largest among EVs, with sizes

(c) removal by distant tissues following entry into systemic circula-

ranging from 50 to 5,000 nm, and contain DNA, RNA, and histone pro-

tion.14 Exosomes carry proteins and nucleic acids from their cell of ori-

teins. Apoptotic bodies are eventually removed by macrophages.6,7

gin,15 and these contents can be delivered to a different recipient cell

Microvesicles (50–1,000 nm) are generally smaller than apoptotic

leading to intercellular communication that can impact different physio-

bodies and are also known as shedding vesicles or exovesicles. Exo-

logical process.5,16,17 Western blotting18–24 and fluorescence-activated

somes (30–120 nm; Figure 1),8 are the smallest among secreted

cell sorting analysis22,25–27 of beads coated with exosomes have helped

vesicles, and consist of a lipid bilayer membrane that surrounds cytosol

determine the presence of known cellular proteins in exosome prepara-

and other contents. Exosomes derived from human embryonic kidney

tions from different cells. In addition, mass spectrometry can be

cells have been observed to shrink in size when stored under 48C tem-

employed

9

to 27–30

identify

unknown

cellular

proteins

present

in

peratures. Exosomes typically do not contain cellular organelles but

exosomes.

can carry different molecular constituents, including proteins and

growth, division, and apoptosis;31,32 changes in gene expression in

Exosome-facilitated messages can regulate cellular

*These authors contributed equally to this study.

C 2017 The Authors. Bioengineering & Translational Medicine is published by Wiley Periodicals, Inc. on behalf of The American Institute of Chemical Engineers V

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

70

|

wileyonlinelibrary.com/journal/btm2

Bioengineering & Translational Medicine 2017; 2: 70–80

INAMDAR

|

ET AL.

71

and clinical medicine. Differential ultracentrifugation remains one of the most common techniques for exosome isolation49–51 and consists of several centrifugation steps that remove cells, large vesicles, and debris at lower centrifugation speeds. The pellets are discarded while the supernatant is subjected to higher centrifugation speeds in order to obtain exosomes as a pellet. Differential ultracentrifugation is commonly employed for the isolation of exosomes but the efficiency of the method is lower when plasma and serum are used due to higher viscosities of these fluids.24,47,52 Density gradient centrifugation combines differential ultracentrifugation with a sucrose density gradient. This method is mainly used for separating exosomes from nonvesicular particles including proteins and protein/RNA aggregates. The method allows separation of low-density exosomes from other contaminants but is highly sensitive to the centrifugation time.24,52,53 Schematic showing the release of exosomes and other vesicles from cells. Adapted from Ref. 8

FIGURE 1

Specific binding of antibodies to receptors present on the surface of exosome have been explored for isolating these vesicles from mixtures54,55; in many cases isolated exosomes are subsequently analyzed

recipient cells can be induced following delivery of multiple miRNAs by exosomes.33 For example, exosomes derived from three different melanoma cell lines, while being morphologically very similar, had different effects on T-cell proliferation/growth suppression.34 In this review, we first discuss cell sources that have been explored for obtaining exosomes and isolation methods that have been employed for obtaining enriched populations of these vesicles. We also discuss the use of exosomes as therapeutic carriers, disease biomarkers, and vaccines.

for DNA or total RNA.56–60 As an application of this approach, antibodies are displayed onto magnetic beads to facilitate the specific binding and isolation of exosomes.61,62 The advantage of this immunoaffinity technique is the presence of various tetraspanins on exosomes isolated from different cell types which can be targeted using antibodies. The immunoaffinity technique employed was reported to be more effective at isolating exosomes compated to ultracentrifugation and density gradient methods.53,63 This method can be extremely effective for smallscale applications with low volume samples but the costs associated

2 | CELL SOURCES FOR EXOSOMES

with scaling up this approach may be prohibitive for large scale isolation procedures.

Cell lines including bEND3 and EL-4 are predominantly used for isola-

Ultrafiltration has been investigated for the separation of exosomes

tion of exosomes used in drug delivery due to the ease of their avail-

from proteins,39,64,65 although the efficacy of this method has not been

ability, the ability to scale up and the ability to generate exosomes of

fully established for clinical samples. A porous membrane can be used

similar quality.35,36 Mesenchymal stem cells or hESC-MSCs are also

for trapping exosomes resulting in their isolation from cell culture media.

excellent sources for isolation of exosomes,37 and it has been observed

The filtration membrane helps concentrate the exosomal population.

that exosomes isolated from these are tolerated well by the immune

Recovery of exosomes from the membranes is facilitated by using etha-

system thereby making them an excellent choice for drug deliv-

nol or NaOH, which is typically followed by rinsing with phosphate-

ery.15,38,39 These cell lines can also be used to isolate exosomes for the

buffered saline.24 However, application of large external force in this

purpose of developing anti-cancer vaccines. It is possible that primary

approach can result in deformation or damage of exosomes.

cells isolated from patients/mice for developing these exosome-based

Polymer-facilitated precipitation, most commonly using polyethyl-

anti-cancer vaccines might demonstrate a batch to batch variation in

ene glycol, is also employed for recovering exosomes from mixtures

strength of the immune response.20,40 The use of exosomes for identi-

(Figure 2).66,67 The main advantage of this method is the use of neutral

fying biomarker levels mandates that patient-derived cells/body fluids

pH. Although, there are no adverse effects on the isolated exosomes,

are used. The presence/absence of disease can be determined by com-

contamination of exosomes with other nonexosomal materials is a sig-

paring exosomal biomarker levels of the patient with that of healthy

nificant drawback of this method.68 In addition, the presence of the

controls.41–45

polymer may interfere with downstream analyses and/or usage.69 Polymer-facilitated precipitation can be significantly improved by using

3 | ISOLATION AND PURIFICATION OF EXOSOMES

methods for removing the polymer used in the operation. Sizeexclusion chromatography is commonly used to separate macromolecules based on size,70 and has also been investigated for exosome

Several methods have been investigated for the isolation and purifica46,47

isolation (Figure 3).71,72 Exosomes isolated using this method are sub-

Centrifugation, filtration,

jected to minimal shear force, resulting in relatively low damage to the

immunological separation, microfluidic isolation,48 and size-exclusion

structure of these vesicles; however, deformation of larger vesicles has

chromatography can be effectively applied in both laboratory research

been reported.73

tion of exosomes from biological fluids.

72

|

FIGURE 2

INAMDAR

ET AL.

Schematic of the polymer-based precipitation method used for the isolation of exosomes. Adapted from Ref. 54

Exosomes isolated using ultracentrifugation—for example, using

tions. Yang et al. hypothesized that exosomes derived from brain cells

the ExoQuick approach—can result in increased yields compared to

displayed brain-specific surface proteins which allowed them to pass

other methods, while also maintaining the quality of the exosomes

through the blood-brain barrier (BBB) and deliver drugs across this bar-

isolated.24 The swift isolation and higher exosome recovery enables

rier.35 Exosomes were isolated from four different cell lines including

analysis of protein expression in the recovered exosomes. Ultracentri-

brain endothelial cell line bEND.3, human glioblastoma cell line U-

fugation with density gradient centrifugation can improve the efficacy

87 MG, human brain neuroectodermal cell line PFSK-1, and human

of the exosome purification without damaging the morphology. Varia-

brain glioblastoma A-172 cells. Rhodamine-123 (2 mg/ml), doxorubicin,

tions in sizes of isolated exosomes have also been observed between

or paclitaxel were independently loaded into different exosomes by

fresh samples and those stored in DMSO and freezing also resulted in

mixing followed by incubation for 2 hr. Doxorubicin-loaded exosomes

degradation of exosomal RNA over time.74

demonstrated higher efficacies for inducing death in U87-MG glioblastoma cells compared to paclitaxel-loaded exosomes. Exosomes isolated

4 | APPLICATIONS OF EXOSOMES

from bEND.3 cells were able to deliver the rhodamine-123 dye across the BBB following delivery via the cardinal vein of zebrafish embryos.

4.1 | Drug and nucleic acid delivery The small sizes of exosomes make them attractive as vehicles in drug and nucleic acid delivery, although detailed molecular characterization of exosomes is necessary before adopting them in widespread applica-

Exosomes from bEND.3 cells were able to deliver drugs to a U-87 MG tumor grown in the brain of zebrafish and were also observed to inhibit VEGF (vascular endothelial growth factor) levels in vivo (Figure 4).35 This, in turn, was able to induce a significant decrease in the tumor size, compared to treatments with the free drug. Intranasal delivery of curcumin or JSI-124 (cucurbitacin I) inhibitor-loaded exosomes was investigated as a potential therapeutic approach for brain inflammatory diseases.75 Three different mouse models exhibiting lipopolysaccharide (LPS)-induced brain inflammation, autoimmune encephalatis, or the GL26 brain tumor model which exhibits inflammation due to infiltration of immune cells including macrophages and T-cells, were used in the study.76 Exosomes were loaded by mixing curcumin with EL-4 (mouse lymphoma cell)-derived exosomes at a temperature of 228C after which the loaded exosomes were separated using sucrose gradient centrifugation. Intranasally delivered exosomes were taken up by microglial cells, which are key mediators in neuro-inflammatory diseases; delivery of curcumin-loaded exosomes resulted in a reduction in activated microglial cells in both encephelatis and LPS-induced brain inflamma-

Schematic of the size-exclusion chromatography approach employed for the isolation and purification of exosomes. Adapted from Ref. 58

FIGURE 3

tion models. Intranasal delivery of exosomes loaded with the STAT3 inhibitor JSI-124 resulted in increased survival of mice with GL26 brain tumors. This study suggests that intranasal delivery of exosome-

INAMDAR

|

ET AL.

73

the loaded vesicles were employed to investigate the knock down of GAPDH gene in C2C12 (murine muscle) and Neuro2A (neuronal cells). Gene knockdown efficacy using exosomes loaded with GAPDH siRNA was similar to that observed using lipofectamine. Exosomes were also well tolerated and did not induce strong immune responses in C57BL/ 6 and BALB/C mice. RVG exosomes were also able to deliver BACE1 siRNA across the BBB.80 Exosomes derived from human bone marrow mesenchymal stem cells were investigated for delivering functional anti-miR-9 to glioblastoma multiforme cells81; communication between MSCs and brain glioblastoma cells can be mediated by exosomes.82 Flow cytometry and quantitative PCR (qPCR) indicated that MSC-derived exosomes, loaded with anti-mir9, led to the reduction of MDR1 expression in T98G and U87 glioblastoma cells. Delivery of anti-mir9 increased the sensitivity of these cells towards temozolomide, resulting in increased cancer cell death.81 In vivo delivery of doxorubicin (Dox)-loaded exosomes across the blood brain barrier in zebrafish model. Significant inhibition of VEGF was observed in Dox-loaded exosomes compared to untreated controls and those treated with unencapsulated doxorubicin. Adapted from Ref. 35

FIGURE 4

encapsulated drugs could lead to a noninvasive approach for direct drug delivery to the CNS.75 Exosomes isolated from RAW264.7 macrophages were loaded with catalase as a potential therapeutic approach for Parkinson’s disease; catalase was loaded in order to potentially degrade reactive oxygen species in order to protect from inflammation.77 Exosomes were loaded using four different methods—incubation with saponin, sonication, freeze-thaw, and extrusion. Sonication-loaded exosomes resulted in the highest uptake of catalase in PC12 neuronal cells in vitro as observed using fluorescence spectroscopy and confocal microscopy. Sonication was carried out using 20% power at 500V, 2kHz for 6 cycles pulsed for 4 s on and paused for 2 s. Extrusion was also employed for loading, and was performed by mixing catalase with exosome solution followed by 10 rounds of extrusion through a Avanti 78

Lipids extruder.

Catalase-loaded exosomes, named exoCAT, pro-

Strategies for loading molecular cargo in exosomes and related efficacies differ based on the chemistry of the loaded molecule. The most common method for loading small-molecule drugs involves mixing the drug solution with a suspension of exosomes and incubating them at 25–378C. The drug loading efficiency is typically determined using liquid chromatrography.83,84 Loading of DNA onto exosomes using electroporation methods is likely restricted by the size of the exosomes with only large exosomes being capable of carrying large linear/plasmid DNA85 The loading efficiencies achieved using electroporation varied between 15 and 30% of those achieved with small molecule drugs and siRNA,86,87 Momen-Heravi et al. were able to achieve up to 55% loading efficiency using electroporation for miRNA molecules.88 Smyth et al. achieved doxorubicin loading efficiencies of 5% by weight of exosomes using the mixing technique.89 Similarly, Sun et al. observed a binding capacity of 2.9 g of curcumin for every gram of exosome using the mixing technique followed by sucrose density gradient centrifugation.36 Kim et al. compared different techniques that is, mixing incubation, electroporation, and sonication for loading the drug paclitaxel into exosomes and observed 1.5, 5, and 29% loading

tected neuronal cells from oxidative stress in vivo following intracranial

efficiencies, respectively.90 In comparison, Yang et al. were able

injection into C57BL/6 mice. Biodistribution of exoCAT following intra-

achieve 5% loading efficiency of siRNA in cationic lipopolymers with

cranial injections indicated localization primarily in neuronal and micro-

an encapsulation efficiency of 70%.91 and Cao et al. obtained 33%

glial cells but also in astrocytes and endothelial cells.

loading efficiency in calcium phosphate nanoparticles.92

Exosomes extracted from cow milk were employed for the delivery

Taken together, these studies indicate the potential utility of exo-

Exosomes

somes in drug and nucleic acid delivery. The choice of cells from which

loaded with withaferin-A were injected intraperitoneally into female

exosomes are isolated, yield of exosome vesicles, cargo loading proce-

athymic nude mice subcutaneously injected with A549 cells. A tumor

dures, selection of targeting biomolecules (e.g., peptides) on the sur-

inhibitory effect was observed with withaferin-A loaded exosomes at

face, biodistribution, and immune response are key factors for

doses lower than those observed with the unencapsulated drug.

consideration for exosome-mediated delivery in future translational

79

of therapeutic molecules against lung and breast cancer.

Exosomes isolated from dendritic cells from the bone marrow of C57BL/6 mice were employed for delivering small interfering RNA

applications. Loading of small-molecule drugs can be efficient, although there is room for improvement in case of loading DNA/siRNA.

(siRNA) to the brain. A Rabies viral glycoprotein (RVG) peptide (single letter

amino

acid

sequence:

YTIWMPENPRPGTPCDIFTNSRGK-

5 | BIOMARKERS

RASNG) was displayed onto the exosomal surface for targeting the acetylcholine receptor in the brain. Electroporation at 400 V and 125

Exosomes play a vital role in cell-cell communication by directly engag-

lF was used to load these exosomes with siRNA against GAPDH, and

ing with surface ligands and/or by transferring their contents between

74

|

INAMDAR

ET AL.

miRNA biomarker expression levels in tumor and exosome-derived samples showing comparable levels of miRNA between the two sample types. Adapted from Ref. 104

FIGURE 5

cells.93–96 Presence of exosomal RNA was implicated as evidence for

culating tumor-derived exosomes derived from lung adenocarcinoma

horizontal transfer of genetic information between various cell

patients were similar to that seen in primary tumors, indicating the

types.97–99 Exosomes are thought to also transfer cellular mRNA as

potential use of these tumor-derived exosomes as biomarkers. In the

well as microRNA which indicates that tumor exosomes are functional

future, it may be possible to analyze miRNA from circulating exosomes

and could suppress mRNA that codes for signal transduction compo-

obviating the need for, or at least complementing, tumor biopsy sam-

100,101

nents within T-cells.

The RNA population in tumor-secreted exo-

somes includes microRNA and it is possible that this exosomal

ples10 in applications related to detecting disease, monitoring response to therapy, and investigating cancer recurrence.

microRNA reflects the parental tumor signature. As a result, microRNA

The mRNA expression of two distinct biomarkers, PCA-3 and

expression profiling can be useful as a diagnostic tool in diseases,

TMPRSS2, is found in exosomes and can be used to provide a direct

including some cancers which lack definitive molecular biomarkers.

link to the incidence of prostate cancer.106 Proteins extracted from uri-

Exosome levels can be slightly elevated in benign tumors and

nary exosomes can be of potential use in diagnostics of urinary tract

highly elevated in cancerous patients as compared to normal controls;

diseases,45 and prostate and bladder cancers. Eight urinary exosomal

exosomes were isolated from the sera of patients/control subjects

proteins were identified as biomarkers when patients with prostate,

using magnetic activated cell sorting. Approximately, 175 different

bladder cancer cells, and healthy cells were compared.107 It has been

miRNA were found to be similar between tumor cells and exosomes.

reported in many cases that exosomal miRNA are prospective bio-

The up-regulated miRNA profile from exosomes also matched up-

markers for renal fibrosis and cardiovascular diseases.108–110

regulated miRNA profiles in ovarian cancer patients at different stages

Potential advantages of using exosomes as biomarkers includes

of the disease. However, this approach was not able to distinguish

the ability to reduce the use of invasive surgery for monitoring disease.

between different stages of cancer.102–105 Expression of miRNA in cir-

Exosomes can be isolated from serum samples and have been

INAMDAR

|

ET AL.

FIGURE 6

75

miRNA profile comparison between tumor and circulating exosomes. Adapted from Ref. 104

successfully used for biomarker detection in ovarian, lung and pancre-

noma antigens in DEXs were used to prime cytotoxic T lymphocytes in

atic cancers.111–113 Exosomal miRNA has also been found to be useful

order to elicit an immune response through the Mart1 specific pathway

in distinguishing between pancreatic carcinomas from benign pancre-

in patients with high-grade melanoma.21,120 In a different study, exo-

113

atic tumors and chronic pancreatitis

and in detection of miRNA

somes were isolated from four different pancreatic cancer cell lines,

(miR-34A) responsible for conferring drug resistance to prostate can-

SOJ-6, BxPC-3, MiaPaCa-2, and Panc-1. Exosomes derived from SOJ-6

41

cer.

Exosomal miRNA appear to be stable and can undergo multiple

and BxPC-3 cells were more effective at reducing growth human pan-

freeze-thaw cycles, variations in pH and heating without undergoing

creatic adenocarcinoma cells compared to those derived from

degradation or loss of expression levels; the miRNA profile is also

MiaPaCa-2 and Panc-1 cells; regulation of Hes-1 protein via downregu-

113

highly specific to cancer/tumor tissue.

However, a key drawback of

lation

of

the

Notch-1

signaling

pathway

and

activation

of

using exosomes as biomarkers markers is that they cannot determine

mitochondria-dependent apoptosis pathway played a key role in the

the severity of the disease because the miRNA levels can be identical

cell ablation efficacy.121

during different stages of the disease as shown by Taylor et al. for

Dendritic cell-derived exosomes were shown to elicit Natural Killer

104,112

ovarian cancer and Rabinowitz et al. in the case of lung cancer.

(NK) cell responses following intradermal injection in C57BL/6 mice.

The potential of using exosomal markers for clinical diagnostics needs

Exosomes isolated from normal volunteers were primed with mela-

to be further investigated in depth because various exosomal compo-

noma associated antigens MAGE3.A1 and MAGE3.DP04, and were

nents including lipids, proteins and miRNAs can be promising in disease

used as vaccines in stage IIIb and stage IV melanoma patients. The

diagnostics.

treatment resulted in a regression of the cancer; while no changes in cytotoxic T-lymphocyte levels were observed, the level of NK cell

6 | ANTICANCER VACCINES

increased with treatment. However, the NK cells obtained from patients that responded to the DEX treatment were more effective at

Exosomes derived from specific sites in the body can be promising can-

killing K562 cells in culture compared to those obtained from

didates for anti-cancer vaccines since they can present antigens against

nonresponders.115,122

that specific type of cancer. Exosomes can be isolated from three sites

These studies indicate potential applications of exosomes in cancer

of origin ascites (ascite-derived exosomes or AEX), dendritic cells (den-

immunotherapy. However, exosomes generally need mature DCs to

dritic cell-derived exosomes or DEX), and tumors (tumor-derived exo-

elicit a T-cell response, since antigens present on exosomes need to be

114–117

The antigens in these exosomes can elicit an

taken up by the dendritic cells before they can activate T-lymphocytes.

immune response via MHC class I or class II molecules on CD81 or

This can be avoided by the use of adjuvants in some cases, which can

CD41 T cells. These exosomal MHC class I/II molecules on exosomes

allow exosomes to directly prime T-lymphocytes. In addition, the reli-

somes or TEX).

118,119

are likely used for communication with specific cell types.

Mela-

ance on an intact immune system can restrict their use in

76

|

INAMDAR

ET AL.

Interactions of dendritic cell-derived exosomes and tumor-derived exosomes with inflammatory cells. Reproduced with permission from Creative Commons. Adapted from Ref. 117

FIGURE 7

immunocompromised or immunosuppressed patients. However, this

gates and other microvascular particles. Optimization of ultracentrifu-

approach can elicit an immune response in already developed cancer

gation coupled with density gradient centrifugation may offer one

which can induce regression of the tumor.122 This approach may also

route toward obtaining enriched populations of exosomes. Further

find less resistance from the suppressive nature of the tumor

advancements that enhance the loading of drug/nucleic acid are neces-

microenvironment,116,117,122–126 which can be a significant advantage.

sary for effective delivery of these therapeutic molecules. Improvements in analytical methods and advances in biomarker discovery can facilitate the use of exosomes in disease detection.

7 | FUTURE DIRECTIONS The potential for exosomes in the field of drug delivery is significant

8 | CONCLUSIONS

owing to their ability to selectively express proteins like tetraspanins which may allow for cell targeting. It will be necessary to obtain highly

Exosomes are starting to gather attention in cancer therapeutics and

pure formulations of exosomes with low amounts of protein aggre-

diagnostics, with several applications in drug delivery, tumor

INAMDAR

ET AL.

immunotherapy, and diagnostic biomarkers. Their unique strengths include enhanced passive targeting due to small size, indigenous nature, and the ability to cross biological barriers. However, the cumbersome nature of the methods required for isolation/purification, inability to distinguish between different cancer stages, and incomplete understanding of their impact on the immune system are some of the current limitations with this technology. It is anticipated that sophisticated engineering and detailed clinical studies that address these limitations will lead to the translation of exosome-based technologies in the future.

|

77

[17] Schorey JS, Bhatnagar S. Exosome function: from tumor immunology to pathogen biology. Traffic. 2008;9(6):871–881. [18] Raposo G, Nijman HW, Stoorvogel W, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 1996;183(3): 1161–1172. [19] Raposo G, Tenza D, Mecheri S, Peronet R, Bonnerot C, Desaymard C. Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation. Mol Biol Cell. 1997;8(12):2631–2645. [20] Zitvogel L, Regnault A, Lozier A, et al. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell derived exosomes. Nat Med. 1998;4(5):594–600. [21] Wolfers J, Lozier A, Raposo G, et al. Nat Med. 2001;7(3):297–303.

L I T E RAT URE CI TE D [1] Walther W, Stein U. Viral vectors for gene transfer. Drugs. 2000; 60 (2):249–271. [2] Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial innovation. Nature. 2000;405(6784):299–304. [3] El Andaloussi S, Mager I, Breakefield XO, Wood MJA. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov. 2013;12(5):347–357. [4] Kooijmans SAA, Vader P, van Dommelen SM, van Solinge WW, Schiffelers RM. Exosome mimetics: a novel class of drug delivery systems. Int J Nanomedicine. 2012;7:1525–1541. [5] Lee Y, EL Andaloussi S, Wood MJA. Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy. Hum Mol Genet. 2012;21(R1):R125–R134. [6] Schiller M, Bekeredjian-Ding I, Heyder P, Blank N, Ho AD, Lorenz HM. Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis. Cell Death Differ. 2008;15(1):183–191. [7] Simpson RJ, Jensen SS, Lim JWE. Proteomic profiling of exosomes: current perspectives. Proteomics. 2008;8(19):4083–4099. [8] Lin J, Li J, Huang B, et al. Exosomes: novel biomarkers for clinical diagnosis. Sci World J. 2015;2015:8. [9] Sokolova V, Ludwig A-K, Hornung S, et al. Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B: Biointerfaces. 2011;87(1):146–150. [10] van den Boorn JG, Daßler J, Coch C, Schlee M, Hartmann G. Exosomes as nucleic acid nanocarriers. Adv Drug Deliv Rev. 2013;65(3): 331–335. [11] Simons M, Raposo G. Exosomes – vesicular carriers for intercellular communication. Curr Opin Cell Biol. 2009;21(4):575–581. ~oz KD, Mahal LK. [12] Batista BS, Eng WS, Pilobello KT, Hendricks-Mun Identification of a conserved glycan signature for microvesicles. J Proteome Res. 2011;10(10):4624–4633.

[22] Blanchard N, Lankar D, Faure F, et al. TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/f complex. J Immunol. 2002;168(7):3235–3241. [23] Escola J-M, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie O, Geuze HJ. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J Biol Chem. 1998;273(32):20121–20127. [24] Lobb RJ, Becker M, Wen Wen S, Wong CSF, Wiegmans AP, Leim€ ller A. Optimized exosome isolation protocol for cell gruber A, Mo culture supernatant and human plasma. J Extracell Vesicles. 2015;4: 27031. [25] Rabesandratana H, Toutant J-P, Reggio H, Vidal M. Decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59) are released within exosomes during in vitro maturation of reticulocytes. Blood. 1998;91(7):2573–2580. [26] Clayton A, Court J, Navabi H, et al. Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods. 2001;247(1-2): 163–174. ry C, Boussac M, Ve ron P, Ricciardi-Castagnoli P, Raposo G, [27] The Garin J, Amigorena S. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol. 2001;166(12):7309–7318. [28] Skokos D, Le Panse S, Villa I, et al. Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes. J Immunol. 2001;166(2):868–876. [29] Van Niel G, Raposo G, Candalh C, et al. Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology. 2001;121(2):337–349. ry C, Regnault A, Garin J, et al. Molecular characterization of [30] The dendritic cell-derived exosomes. J Cell Biol. 1999;147(3):599–610. [31] Azmi AS, Bao B, Sarkar FH. Exosomes in cancer development, metastasis and drug resistance: a comprehensive review. Cancer Metastasis Rev. 2013;32:0–10.

[13] Kalani A, Tyagi A, Tyagi N. Exosomes: mediators of neurodegeneration, neuroprotection and therapeutics. Mol Neurobiol. 2014;49(1): 590–600.

[32] Braicu C, Tomuleasa C, Monroig P, Cucuianu A, Berindan-Neagoe I, Calin GA. Exosomes as divine messengers: are they the Hermes of modern molecular oncology[quest]. Cell Death Differ. 2015;22 (1):34–45.

[14] Pant S, Hilton H, Burczynski ME. The multifaceted exosome: biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities. Biochem Pharmacol. 2012;83(11):1484–1494.

[33] Vlassov AV, Magdaleno S, Setterquist R, Conrad R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta. 2012; 1820(7):940–948.

[15] Lai RC, Yeo RWY, Tan KH, Lim SK. Exosomes for drug delivery—a novel application for the mesenchymal stem cell. Biotechnol Adv. 2013;31(5):543–551.

[34] Wu Y. Melanoma exosomes deliver a complex biological payload that upregulates PTPN11 to suppress T lymphocyte function. Pigment Cell Melanoma Res. 2017;30:203-218.

[16] van Niel G, Porto-Carreiro I, Simoes S, Raposo G. Exosomes: a common pathway for a specialized function. J Biochem. 2006;140 (1):13–21.

[35] Yang T, Martin P, Fogarty B, et al. Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio Rerio. Pharm Res. 2015;32(6):2003–2014.

78

|

[36] Sun D, Zhuang X, Xiang X, et al. A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Mol Ther. 2010;18(9):1606–1614. [37] Chen TS, Arslan F, Yin Y, et al. Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs. J Transl Med. 2011; 9:47-47. [38] Timmers L, Lim SK, Hoefer IE, et al. Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction. Stem Cell Res. 2011;6(3):206–214. [39] Arslan F, Lai RC, Smeets MB, et al. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res. 2013;10(3):301–312.  F, Chaput N, Schartz NEC, et al. Exosomes as potent cell[40] Andre free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells. J Immunol. 2004;172(4):2126–2136. [41] Corcoran C, Rani S, O’Driscoll L. miR-34a is an intracellular and exosomal predictive biomarker for response to docetaxel with clinical relevance to prostate cancer progression. Prostate. 2014;74 (13):1320–1334. [42] Chen X, Ba Y, Ma L, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 2008;18:997–1006. [43] Kosaka N, Iguchi H, Ochiya T. Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis. Cancer Sci. 2010;101:2087–2092. €ltmann H. Serum microRNAs as [44] Brase JC, Wuttig D, Kuner R, Su non-invasive biomarkers for cancer. Mol Cancer. 2010;9:1–9. [45] Zhou H, Cheruvanky A, Hu X, et al. Urinary exosomal transcription factors, a new class of biomarkers for renal disease. Kidney Int. 2008;74(5):613–621. [46] Van Deun J, Mestdagh P, Sormunen R, et al. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracellular Vesicles. 2014;3:24858. [47] Momen-Heravi F, Balaj L, Alian S, et al. Current methods for the isolation of extracellular vesicles. Biol Chem. 2013;394:1253. [48] Chen C, Skog J, Hsu C-H, et al. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab Chip. 2010;10(4): 505–511. [49] Escrevente C, Keller S, Altevogt P, Costa J. Interaction and uptake of exosomes by ovarian cancer cells. BMC Cancer. 2011;11:108-108. [50] Jeppesen DK, Nawrocki A, Jensen SG, et al. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors. Proteomics. 2014;14(6): 699–712. [51] Østergaard O, Nielsen CT, Iversen LV, Jacobsen S, Tanassi JT, Heegaard NHH. Quantitative proteome profiling of normal human circulating microparticles. J Proteome Res. 2012;11(4):2154–2163. [52] Greening DW, Xu R, Ji H, Tauro BJ, Simpson RJ. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. In: Posch A, ed. Proteomic Profiling: Methods and Protocols. New York, NY: Springer; 2015:179–209. ry C, Amigorena S, Raposo G, Clayton A. Isolation and charac[53] The terization of exosomes from cell culture supernatants and biological fluids. In: Bonifacino JS, Harford JB, Lippincott-Schwartz J,

INAMDAR

ET AL.

Yamada KM, eds. Current Protocols in Cell Biology. Hoboken, NJ: John Wiley & Sons, Inc.; 2001. [54] Purushothaman A, Bandari SK, Liu J, Mobley JA, Brown EE, Sanderson RD. Fibronectin on the surface of myeloma cell-derived exosomes mediates exosome-cell interactions. J Biol Chem. 2016;291 (4):1652–1663. [55] Estelles A, Sperinde J, Roulon T, et al. Exosome nanovesicles displaying G protein-coupled receptors for drug discovery. Int J Nanomed. 2007;2(4):751–760. [56] van Balkom BWM, Eisele AS, Pegtel DM, Bervoets S, Verhaar MC. Quantitative and qualitative analysis of small RNAs in human endothelial cells and exosomes provides insights into localized RNA processing, degradation and sorting. J Extracell Vesicles. 2015; 4:26760 [57] Li M, Zeringer E, Barta T, Schageman J, Cheng A, Vlassov AV. Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers. Philos Trans R Soc B Biol Sci. 2014;369(1652):20130502. [58] Guescini M, Genedani S, Stocchi V, Agnati LF. Astrocytes and glioblastoma cells release exosomes carrying mtDNA. J Neural Transm (Vienna). 2009;117(1):1–4. [59] Balaj L, Lessard R, Dai L, et al. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat Comms. 2011;2:180. [60] Thakur BK, Zhang H, Becker A, et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 2014; 24(6):766–769. [61] Pugholm LH, Revenfeld ALS, Søndergaard EKL, Jørgensen MM. Antibody-based assays for phenotyping of extracellular vesicles. BioMed Res Int. 2015;2015:524817. [62] Zarovni N, Corrado A, Guazzi P, et al. Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches. Methods. 2015;87:46–58. [63] Mincheva-Nilsson L, Baranov V, Nagaeva O, Dehlin E. Isolation and characterization of exosomes from cultures of tissue explants and cell lines. In: Colgan JE, Bierer BE, Margulies DH, Shevach EM, Strober W, eds. Current Protocols in Immunology. Hoboken, NJ: John Wiley & Sons, Inc.; 2001. [64] Nordin JZ, Lee Y, Vader P, et al. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine. 2015;11(4):879–883. [65] Gercel-Taylor C, Atay S, Tullis RH, Kesimer M, Taylor DD. Nanoparticle analysis of circulating cell-derived vesicles in ovarian cancer patients. Anal Biochem. 2012;428(1):44–53. [66] Rider MA, Hurwitz SN, Meckes DG. ExtraPEG: a polyethylene glycol-based method for enrichment of extracellular vesicles. Sci Rep. 2016;6:23978. [67] Davies RT, Kim J, Jang SC, Choi E-J, Gho YS, Park J. Microfluidic filtration system to isolate extracellular vesicles from blood. Lab Chip. 2012;12(24):5202–5210. [68] Taylor DD, Zacharias W, Gercel-Taylor C. Exosome isolation for proteomic analyses and RNA profiling. In Simpson JR, Greening WD, eds. Serum/Plasma Proteomics: Methods and Protocols. Totowa, NJ: Humana Press; 2011:235-246. [69] Taylor DD, Shah S. Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods. 2015;87: 3–10. [70] Hong P, Koza S, Bouvier ESP. Size-exclusion chromatography for the analysis of protein biotherapeutics and their

INAMDAR

ET AL.

aggregates. J Liquid Chromatogr Relat Technol. 2012;35(20):2923– 2950. [71] Wang Z, Wu H-j, Fine D, et al. Ciliated micropillars for the microfluidic-based isolation of nanoscale lipid vesicles. Lab Chip. 2013;13(15):2879–2882. di Z, et al. Isolation of exosomes from [72] Baranyai T, Herczeg K, Ono blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PloS One. 2015;10(12):e0145686. [73] Witwer KW, Buzas EI, Bemis LT, et al., Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013;2:20360. doi:10.3402/jev. v2i0.20360

|

79

[88] Momen-Heravi F, Bala S, Bukong T, Szabo G. Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages. Nanomedicine. 2014;10(7):1517–1527. [89] Smyth T, Kullberg M, Malik N, Smith-Jones P, Graner MW, Anchordoquy TJ. Biodistribution and delivery efficiency of unmodified tumor-derived exosomes. J Controlled Release. 2015;199: 145–155. [90] Kim MS, Haney MJ, Zhao Y, et al. Development of exosomeencapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine. 2016;12(3):655–664. [91] Yang X-Z, Dou S, Sun T-M, Mao C-Q, Wang H-X, Wang J. Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy. J Control Release. 2011;156(2):203–211.

[74] Wu Y, Deng W, Klinke DJ. Exosomes: improved methods to characterize their morphology, RNA content, and surface protein biomarkers. Analyst. 2015;140(19):6631–6642.

[92] Cao X, Deng W, Wei Y, et al. Encapsulation of plasmid DNA in calcium phosphate nanoparticles: stem cell uptake and gene transfer efficiency. Int J Nanomed. 2011;6:3335–3349.

[75] Zhuang X, Xiang X, Grizzle W, et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated antiinflammatory drugs from the nasal region to the brain. Mol Ther. 2011;19(10):1769–1779.

[93] Polgar J, Matuskova J, Wagner DD. The P-selectin, tissue factor, coagulation triad. J Thromb Haemost. 2005;3(8):1590–1596.

[76] Candolfi M, Curtin JF, Stephen Nichols W, et al. Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression. J Neurooncol. 2007;85(2): 133–148. [77] Haney MJ, Klyachko NL, Zhao Y, et al. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. J Control Release. 2015; 207:18–30. [78] Haney MJ, Klyachko NL, Zhao Y, et al. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. J Controlled Release. 2015; 207:18–30. [79] Munagala R, Aqil F, Jeyabalan J, Gupta RC. Bovine milk-derived exosomes for drug delivery. Cancer Lett. 2016;371(1):48–61. [80] Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJA. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011;29(4):341–345. [81] Munoz JL, Bliss SA, Greco SJ, Ramkissoon SH, Ligon KL, Rameshwar P. Delivery of functional anti-miR-9 by mesenchymal stem cell-derived exosomes to glioblastoma multiforme cells conferred chemosensitivity. Mol Ther Nucleic Acids. 2013;2:e126. [82] Biancone L, Bruno S, Deregibus MC, Tetta C, Camussi G. Therapeutic potential of mesenchymal stem cell-derived microvesicles. Nephrol Dial Transplant. 2012;27(8):3037–3042. [83] Zhuang X, Xiang X, Grizzle W, et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated antiinflammatory drugs from the nasal region to the brain. Mol Ther. 2011;19(10):1769–1779. [84] Jang SC, Kim OY, Yoon CM, et al. Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors. ACS Nano. 2013;7(9):7698–7710. [85] Lamichhane TN, Raiker RS, Jay SM. Exogenous DNA loading into extracellular vesicles via electroporation is size-dependent and enables limited gene delivery. Mol Pharmaceutics. 2015;12(10): 3650–3657. [86] Banizs AB, Huang T, Dryden K, Berr SS, Stone JR, Nakamoto RK, Shi W, He J. In vitro evaluation of endothelial exosomes as carriers for small interfering ribonucleic acid delivery. Int J Nanomed. 2014; 9:4223–4230. [87] Tian Y, Li S, Song J, et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials. 2014;35(7):2383–2390.

 D, Savani RC, FitzGerald GA. Modulation of [94] Barry OP, Pratico monocyte-endothelial cell interactions by platelet microparticles. J Clin Invest. 1998;102(1):136–144. [95] Rozmyslowicz T, Majka M, Kijowski J, et al. Platelet-and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4HIV. Aids. 2003;17(1):33–42. [96] Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int. 2010;78(9):838–848. [97] Baj-Krzyworzeka M, Szatanek R, We R glarczyk K, et al. Tumourderived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes. Cancer Immunol Immunother. 2006;55(7):808–818. [98] Deregibus MC, Cantaluppi V, Calogero R, et al. Endothelial progenitor cell–derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood. 2007; 110(7):2440–2448. [99] Yuan A, Farber EL, Rapoport AL, et al. Transfer of microRNAs by embryonic stem cell microvesicles. PloS One. 2009;4(3):e4722. [100] Valenti R, Huber V, Filipazzi P, et al. Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-b–mediated suppressive activity on T lymphocytes. Cancer Res. 2006;66(18):9290–9298. [101] Whiteside TL. Immune modulation of T-cell and NK (natural killer) cell activities by TEXs (tumour-derived exosomes). Biochm Soc Trans. 2013;41(1):245–251. [102] Henderson MC, Azorsa DO. The genomic and proteomic content of cancer cell-derived exosomes. Front Oncol. 2012;2:38. [103] Melo SA, Sugimoto H, O’Connell Joyce T, et al. Cancer exosomes perform cell-independent MicroRNA biogenesis and promote tumorigenesis. Cancer Cell. 26(5):707–721. [104] Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumorderived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol. 2008;110(1):13–21. [105] Rosell R, Wei J, Taron M. Circulating MicroRNA signatures of tumor-derived exosomes for early diagnosis of non–small-cell lung cancer. Clin Lung Cancer. 2009;10(1):8–9. [106] Mincheva-Nilsson L, Baranov V. Cancer exosomes and NKG2D receptor–ligand interactions: impairing NKG2D-mediated cytotoxicity and anti-tumour immune surveillance. Semin Cancer Biol. 2014;28:24–30.

80

|

INAMDAR

ET AL.

[107] Smalley DM, Sheman NE, Nelson K, Theodorescu D. Isolation and identification of potential urinary microparticle biomarkers of bladder cancer. J Proteome Res. 2008;7(5):2088–2096.

[117] Kunigelis KE, Graner MW. The dichotomy of tumor exosomes (TEX) in cancer immunity: is it all in the ConTEXt? Vaccines (Basel). 2015;3(4):1019–1051.

[108] Kuwabara Y, Ono K, Horie T, et al. Increased MicroRNA-1 and MicroRNA-133a levels in serum of patients with cardiovascular disease indicate myocardial damage. Circulation. 2011;4(4):446–454.

[118] Lynch S, Santos SG, Campbell EC, et al. Novel MHC class I structures on exosomes. J Immunol. 2009;183(3):1884–1891.

[109] Lv L-L, Cao Y-H, Pan M-M, et al. CD2AP mRNA in urinary exosome as biomarker of kidney disease. Clin Chim Acta. 2014;428:26–31.

[119] LeBrasseur N. MHC and antigen in exosomes. J Cell Biol. 2007; 179(1):4–4.

[110] Chen J-F, Mandel EM, Thomson JM, et al. The role of microRNA1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet. 2006;38(2):228–233.

[120] Escudier B, Dorval T, Chaput N, et al. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derivedexosomes: results of thefirst phase I clinical trial. J Transl Med. 2005;3(1):1.

[111] Resnick KE, Alder H, Hagan JP, Richardson DL, Croce CM, Cohn DE. The detection of differentially expressed microRNAs from the serum of ovarian cancer patients using a novel real-time PCR platform. Gynecol Oncol. 2009;112:55–59.

[121] Ristorcelli E, Beraud E, Mathieu S, Lombardo D, Verine A. Essential role of Notch signaling in apoptosis of human pancreatic tumoral cells mediated by exosomal nanoparticles. Int J Cancer. 2009;125 (5):1016–1026.

[112] Rabinowits G, Gerçel-Taylor C, Day JM, Taylor DD, Kloecker GH. Exosomal MicroRNA: a diagnostic marker for lung cancer. Clin Lung Cancer. 2009;10(1):42–46.

~a H, Seifalian AM. The application of exosomes as [122] Tan A, De La Pen a nanoscale cancer vaccine. Int J Nanomedicine. 2010;5:889–900.

[113] Que R, Ding G, Chen J, Cao L. Analysis of serum exosomal microRNAs and clinicopathologic features of patients with pancreatic adenocarcinoma. World J Surg Onc. 2013;11(1):219. [114] Whiteside TL. Tumor-derived exosomes and their role in tumorinduced immune suppression. Vaccines. 2016;4(4):35.

[123] Simona F, Laura S, Simona T, Riccardo A. Contribution of proteomics to understanding the role of tumor-derived exosomes in cancer progression: state of the art and new perspectives. Proteomics. 2013;13(10-11):1581–1594. € nig A-K, Marme  F, et al. Systemic presence and tumor[124] Keller S, Ko growth promoting effect of ovarian carcinoma released exosomes. Cancer Lett. 2009;278(1):73–81.

[115] Viaud S, Terme M, Flament C, et al. Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Ra. PLoS One. 2009;4(3): e4942.

 F, Schartz NEC, Chaput N, et al. Tumor-derived exosomes: [125] Andre a new source of tumor rejection antigens. Vaccine.2002;20:A28– A31., Supplement 4,

[116] Runz S, Keller S, Rupp C, et al. Malignant ascites-derived exosomes of ovarian carcinoma patients contain CD24 and EpCAM. Gynecol Oncol. 2007;107(3):563–571.

[126] Kharaziha P, Ceder S, Li Q, Panaretakis T. Tumor cell-derived exosomes: a message in a bottle. Biochim Biophys Acta. 2012;1826(1): 103–111.